Draft Method 1633 Analysis of Per- and Polyfluoroalkyl Substances (PFAS) in Aqueous, Solid, Biosolids, and Tissue Samples by LC-MS/MS - Office of ...

Office of Water

 www.epa.gov August 2021

 Draft Method 1633
 Analysis of Per- and Polyfluoroalkyl Substances
 (PFAS) in Aqueous, Solid, Biosolids, and Tissue
 Samples by LC-MS/MS

Draft Method 1633 - subject to revision August 2021

U.S. Environmental Protection Agency
 Office of Water (4303T)
 Office of Science and Technology
 Engineering and Analysis Division
 1200 Pennsylvania Avenue, NW
 Washington, DC 20460

 EPA 821-D-21-001

Draft Method 1633 - subject to revision August 2021

Draft Method 1633
 Analysis of Per- and Polyfluoroalkyl Substances (PFAS) in
Aqueous, Solid, Biosolids, and Tissue Samples by LC-MS/MS

 August 2021

 Notice

 This document represents a draft of a PFAS method currently under development by the EPA Office
 of Water, Engineering and Analysis Division (EAD), in conjunction with the Department of Defense
 (DOD). This method is not required for Clean Water Act compliance monitoring until it has
 been proposed and promulgated through rulemaking.

 A single-laboratory validation of the procedure has been completed and the report on the results of
 that study is being prepared. Historically, EAD posts draft methods on the Clean Water Act website
 after the single-laboratory validation report is completed. However, due to a large number of public
 and stakeholder requests, this method is being posted on the web before the single-laboratory
 validation study report is finalized. A revision of this draft method with a later publication date may
 be issued at that time. No procedural changes are expected as a result of the single-laboratory
 validation, but some of the performance data (which are presented only as examples) may change
 once the statistical analysis of the single-laboratory validation data is completed.

 This draft method has been subjected to multiple levels of review across several EPA Program
 Offices. DOD expects to begin a multi-laboratory validation study of the procedure in late 2021, in
 collaboration with the Office of Water and the Office of Land and Emergency Management.

 The Office of Water will use the results of the multi-laboratory validation study to finalize the method
 and add formal performance criteria. The method validation process may eliminate some of the
 parameters listed in this draft method.

 In the meantime, the Office of Water is releasing this draft on its web site. Laboratories, regulatory
 authorities, and other interested parties are encouraged to review the method, and where appropriate,
 utilize it for their own purposes, with the explicit understanding that this is a draft method, subject to
 revision.

Draft Method 1633 - subject to revision August 2021

Acknowledgements

This draft method was prepared under the direction of Adrian Hanley of the Engineering and Analysis
Division, Office of Science and Technology, within EPA’s Office of Water, in collaboration with the
Department of Defense.

EPA acknowledges the support of a number of organizations in the development and validation of this
draft method, including the developers of the original procedure, the Department of Defense, the
members of EPA’s workgroup, and EPA’s support contractor staff at General Dynamics Information
Technology, including:

 Adrian Hanley EPA Office of Water, Office of Science and Technology, Engineering and Analysis Division
 S. Bekah Burket EPA Office of Water, Office of Science and Technology, Engineering and Analysis Division
 Troy Strock EPA Office of Land and Emergency Management
 Diane Reese EPA Region 4
 Steve Reimer EPA Region 10
 Janice Willey Department of Defense, Naval Sea Systems Command
 Richard H. Anderson Department of Defense, Air Force Civil Engineer Center
 Allyson Buytendyk Institute for Defense Analysis
 Coreen Hamilton SGS-AXYS Analytical
 Ivona Zysk SGS-AXYS Analytical
 Ting Chen SGS-AXYS Analytical
 Henry Huang SGS-AXYS Analytical
 Mirna Alpizar General Dynamics Information Technology
 Harrry McCarty General Dynamics Information Technology

 Disclaimer

See the notice on the title page regarding the status of this method.

Mention of trade names or commercial products does not constitute endorsement or recommendation for
use.

 Contact

Please address questions, comments, or suggestions to:

CWA Methods Team, Engineering and Analysis Division (4303T)
Office of Science and Technology
U.S. Environmental Protection Agency
1200 Pennsylvania Avenue
Washington, DC 20460

https://www.epa.gov/cwa-methods
https://www.epa.gov/cwa-methods/forms/contact-us-about-cwa-analytical-methods

Draft Method 1633 - subject to revision i August 2021

Table of Contents

Acknowledgements ........................................................................................................................................ i
Disclaimer ...................................................................................................................................................... i
Contact ........................................................................................................................................................... i
1.0 Scope and Application ...................................................................................................................... 1
2.0 Summary of Method ......................................................................................................................... 1
3.0 Definitions......................................................................................................................................... 2
4.0 Contamination and interferences ...................................................................................................... 2
5.0 Safety ................................................................................................................................................ 3
6.0 Equipment and Supplies.................................................................................................................... 5
7.0 Reagents and standards ..................................................................................................................... 8
8.0 Sample collection, preservation, storage, and holding times .......................................................... 13
9.0 Quality Control ............................................................................................................................... 14
10.0 Calibration and Standardization ...................................................................................................... 18
11.0 Sample preparation and extraction.................................................................................................. 25
12.0 Extraction, Cleanup, and Concentration ......................................................................................... 30
13.0 Instrumental Analysis ..................................................................................................................... 32
14.0 Performance Tests during Routine Operations ............................................................................... 33
15.0 Data Analysis and Calculations ...................................................................................................... 35
16.0 Method Performance ....................................................................................................................... 38
17.0 Pollution Prevention........................................................................................................................ 38
18.0 Waste Management ......................................................................................................................... 39
19.0 References ....................................................................................................................................... 39
20.0 Tables, Diagrams, Flowcharts, and Validation Data....................................................................... 41
21.0 Glossary .......................................................................................................................................... 55

Appendix A - Sample Pre-screening Instructions ....................................................................................... 59
Appendix B - Aqueous Sample Subsampling Instructions ......................................................................... 60

Draft Method 1633 - subject to revision ii August 2021

DRAFT Method 1633 - Analysis of Per- and Polyfluoroalkyl Substances (PFAS)
 in Aqueous, Solid, Biosolids, and Tissue Samples by LC-MS/MS

1.0 Scope and Application

1.1 Method 1633 is for use in the Clean Water Act (CWA) for the determination of the per- and
 polyfluoroalkyl substances (PFAS) in Table 1 in aqueous, solid (soil, biosolids, sediment) and tissue
 samples by liquid chromatography/mass spectrometry (LC-MS/MS).

1.2 The method calibrates and quantifies PFAS analytes using isotopically labeled standards. Where
 linear and branched isomers are present in the sample and either qualitative or quantitative standards
 containing branched and linear isomers are commercially available, the PFAS analyte is reported as
 a single analyte consisting of the sum of the linear and branched isomer concentrations.

1.3 The instrumental portion of this method is for use only by analysts experienced with LC-MS/MS or
 under the close supervision of such qualified persons. Each laboratory that uses this method must
 demonstrate the ability to generate acceptable results using the procedure in Section 9.2.

1.4 By their very nature, many components of PFAS present analytical challenges unique to this class
 of analytes. For example, PFAS analytes readily adhere to the walls of the sample containers and
 may also stratify in the container. EPA has included procedures in the method that must be
 employed to address such challenges (see Section 11.0 and Appendices A and B).

1.5 This method is “performance-based,” which means that modifications may be made without
 additional EPA review to improve performance (e.g., overcome interferences, or improve the
 sensitivity, accuracy, or precision of the results) provided that all performance criteria in this
 method are met. Requirements for establishing equivalency are in Section 9.1.2 and include
 9.1.2.2c. For CWA uses, additional flexibility is described at 40 CFR 136.6. Changes in
 performance, sensitivity, selectivity, precision, recovery, etc., that result from modifications within
 the scope of 40 CFR Part 136.6, and Section 9.0 of this method must be documented, as well as
 how these modifications compare to the specifications in this method. Changes outside the scope
 of 40 CFR Part 136.6 and Section 9.0 of this method may require prior review or approval.

2.0 Summary of Method

Environmental samples are prepared and extracted using method-specific procedures. Sample extracts
are subjected to cleanup procedures designed to remove interferences. Analyses of the sample extracts
are conducted by LC-MS/MS in the multiple reaction monitoring (MRM) mode. Sample concentrations
are determined by isotope dilution or extracted internal standard quantification (see Section 10.3) using
isotopically labeled compounds added to the samples before extraction.

2.1 Extraction

 2.1.1 Aqueous samples are spiked with isotopically labeled standards, extracted using
 solid-phase extraction (SPE) cartridges and undergo cleanup using carbon before
 analysis.

 2.1.2 Solid samples are spiked with isotopically labeled standards, extracted into basic
 methanol, and cleaned up by carbon and SPE cartridges before analysis.

Draft Method 1633 - subject to revision 1 August 2021

2.1.3 Tissue samples are spiked with isotopically labeled standards, extracted in potassium
 hydroxide and acetonitrile followed by basic methanol, and cleaned up by carbon and SPE
 cartridges before analysis.

2.2 This method measures the analytes as either their anions or neutral forms. The default approach for
 Clean Water Act uses of the method is to report the analytes in their acid or neutral forms, using the
 equations in Section 15.2, although the differences between the anion and acid form concentrations
 are minimal (See Table 8). Other project-specific reporting schemes may be used where required.

2.3 Individual PFAS analytes are identified through peak analysis of the quantification and
 confirmation ions, where applicable.

2.4 Quantitative determination of target analyte concentrations is made with respect to an isotopically
 labeled PFAS standard; the concentrations are then used to convert raw peak areas in sample
 chromatograms to final concentrations.

2.5 Results for target analytes are recovery corrected by the method of quantification (i.e., either
 isotope dilution or extracted internal standard quantification, see Section 10.3). Isotopically labeled
 compound recoveries are determined by comparison to the responses of one of seven non-extracted
 internal standards (a.k.a., the “recovery” standards) and are used as general indicators of overall
 analytical quality.

2.6 The quality of the analysis is assured through reproducible calibration and testing of the extraction,
 cleanup, and LC-MS/MS systems.

3.0 Definitions

 Definitions are provided in the glossary at the end of this method.

4.0 Contamination and interferences

4.1 Solvents, reagents, glassware, and other sample processing hardware may yield artifacts and
 elevated baselines causing misinterpretation of chromatograms. Specific selection of reagents and
 solvents may be required.

4.2 Clean all equipment prior to, and after each use to avoid PFAS cross-contamination. Typical
 cleaning solvents used include water, methanol, and methanolic ammonium hydroxide. The
 residual PFAS content of disposable plasticware and filters must be verified by batch/lot number
 and may be used without cleaning if PFAS levels are less than half the Minimum Level (ML, see
 Table 6).

 4.2.1 All glass equipment that is used in the preparation or storage of reagents is cleaned by
 washing with detergent and baking in a kiln or furnace (Section 6.2.2). After detergent
 washing, glassware should be rinsed immediately with reagent water. Prior to use, baked
 glassware must be solvent rinsed and then air dried. A solvent rinse procedure using
 methanolic ammonium hydroxide (1%), toluene, and methanol is recommended.

 4.2.2 All parts of the SPE manifold must be cleaned between samples by sonicating in
 methanolic ammonium hydroxide (1%) and air drying prior to use. Smaller parts, like the
 needles, adapters, reservoirs, and stopcocks associated with the manifold require rinsing

Draft Method 1633 - subject to revision 2 August 2021

with tap water prior to sonicating in methanolic ammonium hydroxide (1%) and air drying.
 When in use, after loading the samples but prior to elution procedures, the chamber must be
 rinsed with methanolic ammonium hydroxide (1%).

 4.2.3 All equipment used in the filleting, dissecting, shucking, compositing, and homogenization
 of tissue must be cleaned with detergent and hot water, then rinsed with ultra-pure water
 followed by a series of solvent rinses. A typical solvent rinse procedure would be acetone,
 followed by toluene, and then dichloromethane.

4.3 All materials used in the analysis must be demonstrated to be free from interferences by running
 method blanks (Section 9.5) at the beginning and with each sample batch (samples started through
 the extraction process on a given analytical batch to a maximum of 20 field samples).

 4.3.1 The reference matrix must simulate, as closely as possible, the sample matrix being tested.
 Ideally, the reference matrix should not contain PFAS in detectable amounts but should
 contain potential interferents in the concentrations expected to be found in the samples to
 be analyzed.

 4.3.2 For tissue, chicken breast or other similar animal tissue (see Section 7.2.3) may be used as
 the reference matrix. The laboratory must verify that the source product used does not
 contain PFAS in detectable amounts.

 4.3.3 When a reference matrix that simulates the sample matrix under test is not available,
 reagent water (Section 7.2.1) can be used to simulate water samples and Ottawa sand
 and/or reagent-grade sand (Section 7.2.2) can be used to simulate soils.

4.4 Interferences co-extracted from samples will vary considerably from source to source, depending
 on the diversity of the site being sampled. Interfering compounds may be present at concentrations
 several orders of magnitude higher than the native PFAS. Because low levels of PFAS are
 measured by this method, elimination of interferences is essential. The cleanup steps given in
 Section 12.0 can be used to reduce or eliminate these interferences and thereby permit reliable
 determination of the PFAS at the levels shown in Table 6. The most frequently encountered
 interferences are fluoropolymers; however, when analyzing whole fish samples, bile salts (e.g.,
 Taurodeoxycholic Acid [TDCA]) can interfere in the chromatography. For this reason, analysis of
 a standard containing TDCA is required as part of establishing the initial chromatographic
 conditions (see Sections 10.2.2.5 and 10.3.5).

4.5 Each piece of reusable glassware may be numbered to associate that glassware with the processing
 of a particular sample. This may assist the laboratory in tracking possible sources of contamination
 for individual samples, identifying glassware associated with highly contaminated samples that may
 require extra cleaning, and determining when glassware should be discarded.

5.0 Safety

5.1 The toxicity or carcinogenicity of each chemical used in this method has not been precisely
 determined; however, each compound should be treated as a potential health hazard. Exposure to
 these compounds should be reduced to the lowest possible level.

 5.1.1 PFOA has been described as likely to be carcinogenic to humans. Pure standards should be
 handled by trained personnel, with suitable protection to skin and eyes, and care should be
 taken not to breathe the vapors or ingest the materials.

Draft Method 1633 - subject to revision 3 August 2021

5.1.2 It is recommended that the laboratory purchase dilute standard solutions of the analytes in
 this method. However, if primary solutions are prepared, they must be prepared in a hood,
 following universal safety measures.

5.2 The laboratory is responsible for maintaining a current awareness file of Occupational Safety and
 Health Administration (OSHA) regulations regarding the safe handling of the chemicals specified
 in this method. A reference file of safety data sheets (SDS) should also be made available to all
 personnel involved in these analyses. It is also suggested that the laboratory perform personal
 hygiene monitoring of each analyst who uses this method and that the results of this monitoring be
 made available to the analyst. Additional information on laboratory safety can be found in
 References 1-4. The references and bibliography at the end of Reference 3 are particularly
 comprehensive in dealing with the general subject of laboratory safety.

5.3 Samples suspected to contain these compounds are handled using essentially the same techniques
 employed in handling radioactive or infectious materials. Well-ventilated, controlled access
 laboratories are required. Assistance in evaluating the health hazards of particular laboratory
 conditions may be obtained from certain consulting laboratories and from State Departments of
 Health or Labor, many of which have an industrial health service. Each laboratory must develop a
 strict safety program for handling these compounds.

 5.3.1 Facility – When finely divided samples (dusts, soils, dry chemicals) are handled, all
 operations (including removal of samples from sample containers, weighing, transferring,
 and mixing) should be performed in a glove box demonstrated to be leak tight or in a fume
 hood demonstrated to have adequate air flow. Gross losses to the laboratory ventilation
 system must not be allowed. Handling of the dilute solutions normally used in analytical
 and animal work presents no inhalation hazards except in the case of an accident.

 5.3.2 Protective equipment – Disposable plastic gloves, apron or lab coat, safety glasses or mask,
 and a glove box or fume hood adequate for radioactive work should be used. During
 analytical operations that may give rise to aerosols or dusts, personnel should wear
 respirators equipped with activated carbon filters. Eye protection (preferably full-face
 shields) must be worn while working with exposed samples or pure analytical standards.
 Latex gloves are commonly used to reduce exposure of the hands.

 5.3.3 Training – Workers must be trained in the proper method of removing contaminated gloves
 and clothing without contacting the exterior surfaces.

 5.3.4 Personal hygiene – Hands and forearms should be washed thoroughly after each
 manipulation and before breaks (coffee, lunch, and shift).

 5.3.5 Confinement – Isolated work areas posted with signs, segregated glassware and tools, and
 plastic absorbent paper on bench tops will aid in confining contamination.

 5.3.6 Waste Handling – Good technique includes minimizing contaminated waste. Plastic bag
 liners should be used in waste cans. Janitors and other personnel should be trained in the
 safe handling of waste.

 5.3.7 Laundry – Clothing known to be contaminated should be collected in plastic bags. Persons
 that convey the bags and launder the clothing should be advised of the hazard and trained in
 proper handling. The clothing may be put into a washer without contact if the launderer
 knows of the potential problem. The washer should be run through a cycle before being
 used again for other clothing.

Draft Method 1633 - subject to revision 4 August 2021

5.4 Biosolids samples may contain high concentrations of biohazards and must be handled with gloves
 and opened in a fume hood or biological safety cabinet to prevent exposure. Laboratory staff
 should know and observe the safety procedures required in a microbiology laboratory that handles
 pathogenic organisms when handling biosolids samples.

6.0 Equipment and Supplies

Note: Brand names, suppliers, and part numbers are for illustration purposes only and no endorsement
 is implied. Equivalent performance may be achieved using apparatus and materials other than
 those specified here. Meeting the performance requirements of this method is the responsibility
 of the laboratory.

6.1 Sampling equipment for discrete or composite sampling

 6.1.1 Sample bottles and caps

 Note: Do not use PTFE-lined caps on sample containers.

 6.1.1.1 Liquid samples (waters, sludges, and similar materials containing < 50 mg
 solids per sample) – Sample bottle, HDPE, with linerless HDPE or
 polypropylene caps.

 Note: At least two aliquots of aqueous samples are collected to allow sufficient volume
 for the determination of percent solids and for pre-screening analysis. One
 aliquot should be collected in a 500-mL container while the second aliquot may
 be collected in a smaller sample container (e.g., 250-mL or 125-mL).

 6.1.1.2 Solid samples (soils, sediments, and biosolids that contain more than 50 mg
 solids) – Sample bottle or jar, wide-mouth, HDPE, 500-mL, with linerless
 HDPE or polypropylene caps.

 6.1.1.3 Tissue samples – Sample jar, wide-mouth HDPE, 100-mL, with linerless HDPE
 or polypropylene caps.

 6.1.2 Compositing equipment – Automatic or manual compositing system incorporating
 containers cleaned per bottle cleaning procedure above. Only HDPE tubing must be used.
 If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber
 tubing may be used in the pump only. Before use, the tubing must be thoroughly rinsed
 with methanol, followed by repeated rinsing with reagent water to minimize sample
 contamination. An integrating flow meter is used to collect proportional composite
 samples.

6.2 Equipment for glassware cleaning

Note: If blanks from bottles or other glassware, show no detectable PFAS contamination when using
 fewer cleaning steps than required above, unnecessary cleaning steps and equipment may be
 eliminated.

 6.2.1 Laboratory sink with overhead fume hood

Draft Method 1633 - subject to revision 5 August 2021

6.2.2 Kiln – Capable of reaching 450 ºC within 2 hours and maintaining 450 - 500 ºC ± 10 ºC,
 with temperature controller and safety switch (Cress Manufacturing Co., Santa Fe Springs,
 CA, B31H, X31TS, or equivalent). For safety, the kiln or furnace should be vented outside
 the laboratory, or to a trapping system.

6.3 Equipment for sample preparation

 6.3.1 Polyethylene gloves

 6.3.2 Laboratory fume hood (of sufficient size to contain the sample preparation equipment listed
 below)

 6.3.3 Glove box (optional)

 6.3.4 Tissue homogenizer – Pro Scientific PRO400DS homogenizer or equivalent with stainless
 steel macro-shaft and turbo-shear blade

 6.3.5 Meat grinder – Hobart, or equivalent, with 3- to 5-mm holes in inner plate

 6.3.6 Equipment for determining percent moisture

 6.3.6.1 Oven – Capable of maintaining a temperature of 110 ± 5 ºC

 6.3.6.2 Desiccator

 6.3.7 Balances

 6.3.7.1 Analytical – Capable of weighing 0.1 mg

 6.3.7.2 Top loading – Capable of weighing 10 mg

 6.3.8 Aluminum foil

 6.3.9 Disposable spoons, 10 mg, polypropylene or stainless steel

 6.3.10 Ultrasonic mixer (sonicator)

 6.3.11 HDPE bottles, with linerless HDPE or polypropylene caps – 60 mL

 6.3.12 pH Paper, range 0-14 - (Whatman® PanpehaTM or equivalent), 0.5-unit readability

 6.3.13 Analog or digital vortex mixer, single or multi-tube (Fisher Scientific 02-215-452, or
 equivalent)

 6.3.14 Volumetric flasks, Class A

 6.3.15 Disposable polypropylene collection tubes (13 x 100 mm, 8 mL)

 6.3.16 Variable speed mixing table (FisherbrandTM Nutating mixer or equivalent)

Draft Method 1633 - subject to revision 6 August 2021

6.4 Filtration

 6.4.1 Silanized glass wool (Sigma-Aldrich, Cat # 20411 or equivalent) – store in a clean glass jar
 and rinsed with methanol (2 times) prior to use.

 6.4.2 Disposable syringe filter, 25-mm, 0.2-µm Nylon membrane, PALL/Acrodisc or equivalent

 6.4.3 Glass fiber filter, 47 mm, 1 µm, PALL A/E or equivalent

6.5 Centrifuge apparatus

 6.5.1 Centrifuge (Thermo Scientific Legend RT+, 16 cm rotor, or equivalent), capable of
 reaching at least 3000 rpm

 6.5.2 Centrifuge tubes – Disposable polypropylene centrifuge tubes (50 mL)

6.6 Pipettes

 6.6.1 Norm-Ject® syringe (or equivalent), polypropylene/HDPE, 5 mL

 6.6.2 Variable volume pipettes with disposable HDPE or polypropylene tips (10 µL to 5 mL) –
 used for preparation of calibration standards and spiked samples.

 6.6.3 Disposable glass pipets

 6.6.4 Calibrated mechanical pipettes or Hamilton graduated syringes

6.7 Solid-Phase Extraction

 6.7.1 Solid-phase extraction (SPE) cartridges (Waters Oasis WAX 150 mg, Cat # 186002493 or
 equivalent). The SPE sorbent must have a pKa above 8 so that it remains positively
 charged during the extraction.

 Note: SPE cartridges with different bed volume (e.g., 500 mg) may be used; however, the
 laboratory must demonstrate that the bed volume does not negatively affect analyte
 absorption and elution, by performing the initial demonstration of capability analyses
 described in Section 9.2.

 6.7.2 Vacuum manifold for SPE Cartridges (WatersTM extraction manifold #WAT200607 or
 equivalent)

6.8 Evaporation

 6.8.1 Automatic or manual solvent evaporation system (TurboVap® LV or
 equivalent)

 6.8.2 Evaporation/concentrator tubes: 60 mL clear glass vial, 30 x 125 mm, without
 caps (Wheaton Cat # W226060 or equivalent). Cover with foil if required.

Draft Method 1633 - subject to revision 7 August 2021

6.9 Vials

 6.9.1 Snap cap/crimp top vials, 300 µL, polypropylene (12 x 32 mm) – used in sample
 pre-screening (DWK Life Sciences Cat # 225180 or equivalent)

 6.9.2 Polypropylene crimp/snap vials, 1 mL (Agilent Cat # 5182-0567 or equivalent)

 6.9.3 Clear snap cap, PVDC film/white silicone, 11 mm (American Chromatography
 Supplies Cat # C299-11 or equivalent)

 6.9.4 Single step filter vials (Restek Thomson SINGLE StEP® Standard Filter Vials,
 0.2 μm Nylon membrane, with Black Preslit caps Cat # 25891 or equivalent) –
 used in sample pre-screening.

6.10 Instrument

 6.10.1 Ultra high-performance liquid chromatograph (UPLC also called UHPLC) or high-
 performance liquid chromatograph (HPLC) equipped with tandem quadrupole mass
 spectrometer (Waters Xevo TQ-S Micro or equivalent).

 6.10.2 C18 column, 1.7 µm, 50 x 2.1 mm (Waters Acquity UPLC® BEH or equivalent)

 6.10.3 Guard column (Phenomenex Kinetex® Evo C18 or equivalent)

 6.10.4 Trap/delay column (Purospher Star RP-18 endcapped [3 μm] Hibar® RT 50-4 or
 equivalent)

6.11 Bottles, HDPE or glass, with linerless HDPE or polypropylene caps. Various sizes. To store
 prepared reagents.

7.0 Reagents and standards

7.1 Reagents

 Reagents prepared by the laboratory may be stored in either glass or HDPE containers. Proper
 cleaning procedures (Section 4.2) must be followed prior to using the containers.

 7.1.1 Acetic acid - ACS grade or equivalent, store at room temperature

 7.1.2 Acetic acid (0.1%) - dissolve acetic acid (1 mL) in reagent water (1 L), store at room
 temperature, replace after 3 months. This reagent is used only for sample extract dilution.

 7.1.3 Acetonitrile – UPLC grade or equivalent, verified before use, store at room temperature

 7.1.4 Ammonium acetate - (Caledon Ultra LC/MS grade, or equivalent), store at 2-8° C, replace
 2 years after opening date

 7.1.5 Ammonium hydroxide - certified ACS+ grade or equivalent, 30% in water, store at room
 temperature

Draft Method 1633 - subject to revision 8 August 2021

7.1.6 Aqueous ammonium hydroxide (3%) - add ammonium hydroxide (10 mL, 30%) to reagent
 water (90 mL), store at room temperature, replace after 3 months

 7.1.7 Methanolic ammonium hydroxide

 7.1.7.1 Methanolic ammonium hydroxide (0.3%) - add ammonium hydroxide (1 mL,
 30%) to methanol (99 mL), store at room temperature, replace after 1 month

 7.1.7.2 Methanolic ammonium hydroxide (1%) - add ammonium hydroxide (3.3 mL,
 30%) to methanol (97 mL), store at room temperature, replace after 1 month

 7.1.7.3 Methanolic ammonium hydroxide (2%) - add ammonium hydroxide (6.6 mL,
 30%) to methanol (93.4 mL), store at room temperature, replace after 1 month

 7.1.8 Methanolic potassium hydroxide (0.05 M) – add 3.3 g of potassium hydroxide to 1 L of
 methanol, store at room temperature, replace after 3 months

 7.1.9 Methanol with 4% water, 1% ammonium hydroxide and 0.625% acetic acid - add
 ammonium hydroxide (3.3 mL, 30%), reagent water (1.7 mL) and acetic acid (0.625 mL) to
 methanol (92 mL), store at room temperature, replace after 1 month. This solution is used
 to prepare the instrument blank (Section 7.3.6).

 7.1.10 Eluent A – Acetonitrile, Caledon Ultra LCMS grade or equivalent

 7.1.11 Eluent B - 2 mM ammonium acetate in 95:5 water/acetonitrile. Dissolve 0.154 g of
 ammonium acetate (Section 7.1.4) in 950 mL of water and 50 mL of acetonitrile (Caledon
 Ultra LCMS grade, or equivalent). Store at room temperature, shelf life 2 months.

 7.1.12 Formic acid - (greater than 96% purity or equivalent), verified by lot number before use,
 store at room temperature

 7.1.13 Formic acid

 7.1.13.1 Formic acid (aqueous, 0.1 M) - dissolve formic acid (4.6 g) in reagent water (1
 L), store at room temperature, replace after 2 years

 7.1.13.2 Formic acid (aqueous, 0.3 M) - dissolve formic acid (13.8 g) in reagent water (1
 L), store at room temperature, replace after 2 years

 7.1.13.3 Formic acid (aqueous, 5% v/v) - mix 5 mL formic acid with 95 mL reagent
 water, store at room temperature, replace after 2 years

 7.1.13.4 Formic acid (aqueous, 50% v/v) - mix 50 mL formic acid with 50 mL reagent
 water, store at room temperature, replace after 2 years

 7.1.13.5 Formic acid (methanolic 1:1, 0.1 M formic acid/methanol) - mix equal volumes
 of methanol and 0.1 M formic acid, store at room temperature, replace after 2
 years

 7.1.14 Methanol - (HPLC grade or better, 99.9% purity), verified by lot number before use, store
 at room temperature

Draft Method 1633 - subject to revision 9 August 2021

7.1.15 Potassium hydroxide – certified ACS or equivalent, store at room temperature, replace after
 2 years

 7.1.16 Reagent water – Laboratory reagent water, test by lot/batch number for residual PFAS
 content

 7.1.17 Carbon – EnviCarb® 1-M-USP or equivalent, verified by lot number before use, store at
 room temperature. Loose carbon allows for better adsorption of interferent organics.

 Note: The single-laboratory validation laboratory achieved better performance with loose carbon
 than carbon cartridges. Loose carbon will be used for the multi-laboratory validation to
 set statistically based method criteria. Once the method is multi-laboratory validated,
 laboratories will have the flexibility to use carbon cartridges as long as all method QC
 criteria are met.

 7.1.18 Toluene – HPLC grade, verified by lot number before use. Store at room temperature.

 7.1.19 Acetone - Pesticide grade, verified by lot number before use in rinsing tissue dissection and
 processing equipment.

 7.1.20 Dichloromethane (methylene chloride), pesticide grade, verified by lot number before use
 in rinsing tissue dissection and processing equipment.

7.2 Reference matrices – Matrices in which PFAS and interfering compounds are not detected by this
 method. These matrices are to be used to prepare the batch QC samples (e.g., method blank, and
 ongoing precision and recovery sample).

 7.2.1 Reagent water – purified water, Type I

 7.2.2 Solids reference matrix – Ottawa or reagent-grade sand

 7.2.3 Tissue reference matrix – chicken breast or similar animal tissue

7.3 Standard solutions – Prepare from materials of known purity and composition or purchase as
 solutions or mixtures with certification to their purity, concentration, and authenticity. Observe the
 safety precautions in Section 5.

 Purchase of commercial standard solutions or mixtures is highly recommended for this method;
 however, when these are not available, preparation of stock solutions from neat materials may be
 necessary. If the chemical purity is 98% or greater, the weight may be used without correction to
 calculate the concentration of the standard. Dissolve an appropriate amount of assayed reference
 material in the required solvent. For example, weigh 10 to 20 mg of an individual compound to
 three significant figures in a 10-mL ground-glass-stoppered volumetric flask and fill to the mark
 with the required solvent. Once the compound is completely dissolved, transfer the solution to a
 clean vial and cap.

 When not being used, store standard solutions in the dark at less than 4 °C unless the vendor
 recommends otherwise in screw-capped vials with foiled-lined caps. Place a mark on the vial at the
 level of the solution so that solvent loss by evaporation can be detected. Replace the solution if
 solvent loss has occurred.

Draft Method 1633 - subject to revision 10 August 2021

Note: Native PFAS standards are available from several suppliers. Isotopically labeled compounds are
 available from Cambridge Isotope Laboratories and Wellington Laboratories, but may also be
 available from other suppliers. Listing of these suppliers does not constitute a recommendation
 or endorsement for use. All diluted solutions must be stored in glass or HDPE containers that
 have been thoroughly rinsed with methanol.
 18
 O-mass labeled perfluoroalkyl sulfonates may undergo isotopic exchange with water under
 certain conditions, which lowers the isotopic purity of the standards over time.

 The laboratory must maintain records of the certificates for all standards for traceability purposes.
 Copies of the certificates must be provided as part of the data packages in order to check that proper
 calculations were performed.

 7.3.1 Extracted Internal Standard (EIS) – (a.k.a. isotopically labeled compound) Prepare the EIS
 solution containing the isotopically labeled compounds listed in Table 3 as extracted
 internal standards in methanol from prime stocks. An aliquot of EIS solution, typically 50
 µL, is added to each sample prior to extraction. Table 3 presents the nominal amounts of
 EIS compounds added to each sample. The list of isotopically labeled compounds in Table
 3 represents the compounds that were available at the time this method was validated.
 Other isotopically labeled compounds may be used as they become available.

 7.3.2 Non-Extracted Internal Standard (NIS) - The NIS solution containing the isotopically
 labeled compounds listed in Table 3 as non-extracted internal standards is prepared in
 methanol from prime stock. An aliquot of NIS solution, typically 50 µL, is added to each
 sample prior to instrumental analysis. Table 3 presents the nominal amounts of NIS
 compounds added to each sample.

 7.3.3 Native Standards Solution - Prepare a spiking solution, containing the method analytes
 listed in Table 4, in methanol from prime stocks. The solution is used to prepare the
 calibration standards and to spike the known reference QC samples that are analyzed with
 every batch. Quantitative standards containing a mixture of branched and linear isomers
 must be used for method analytes if they are commercially available. Currently, these
 include PFOS, PFHxS, NMeFOSAA, and NEtFOSAA.

 7.3.4 Calibration standard solutions – A series of calibration solutions containing the target
 analytes and the 13C-, 18O-, and deuterium-labeled extracted internal standards (EIS) and
 non-extracted internal standards (NIS) is used to establish the initial calibration of the
 analytical instrument. The concentration of the method analytes in the solutions varies to
 encompass the working range of the instrument, while the concentrations of the EIS and
 NIS remain constant. The calibration solutions are prepared using methanol, methanolic
 ammonium hydroxide (2%), water, acetic acid and the method analyte and isotopically
 labeled compound standard solutions. After dilution, the final solution will match the
 solvent mix of sample extracts, which contain methanol with 4% water, 1% ammonium
 hydroxide and 0.625% acetic acid (Section 7.1.9). Calibration standard solutions do not
 undergo solid phase extraction/cleanup.

 Concentrations for seven calibration solutions are presented in Table 4. A minimum of six
 contiguous calibrations standards are required for a valid analysis when using a linear
 calibration model, with at least five of the six calibration standards being within the
 quantitation range (e.g., from the LOQ to the highest calibration standard). If a second-
 order calibration model is used, then a minimum of seven calibration standards are

Draft Method 1633 - subject to revision 11 August 2021

required, with at least six of the seven calibration standards within the quantitation range.
 The lowest level calibration standard must meet a signal-to-noise ratio of 3:1 and be at a
 concentration less than or equal to the Limit of Quantitation (LOQ). All initial calibration
 requirements listed in Table 7 must be met. An instrument sensitivity check (ISC) standard
 at the concentration of the lowest calibration standard within the quantitation range is
 required to be analyzed at the beginning of the analytical run (Section 10.3.3.1 and Section
 13.3). A mid-level calibration solution is analyzed at least every ten samples or less, on an
 ongoing basis for the purpose of calibration verification. A mid-level calibration
 verification (CV) standard must also be analyzed after all sample analyses in order to
 bracket the analytical batch.

 Note: Additional calibration standards, at levels lower than the lowest calibration standard listed
 in the method, may be added to accommodate a lower limit of quantitation if the instrument
 sensitivity allows. Calibration standards at the high end of the calibration may be
 eliminated if the linearity of the instrument is exceeded or at the low end if those
 calibration standards do not meet the S/N ratio criterion of 3:1, as long as the required
 number of calibration points is met. All analytes with commercially available stable
 isotope analogues must be quantified using isotope dilution.

 7.3.5 Qualitative Standards - Standards that contain mixtures of the branched and linear isomers
 of the method analytes and that are used for comparison against suspected branched isomer
 peaks in field samples. These qualitative standards are not required for those analytes
 where the quantitative standards in Section 7.3.3 already contain the branched and linear
 isomers. Qualitative standards that are currently commercially available include PFOA,
 PFNA, PFOSA, NMeFOSA, NEtFOSA, NEtFOSE, and NMeFOSE.

 7.3.6 Instrument Blank – During the analysis of a batch of samples, a solvent blank is analyzed
 after samples containing high level of target compounds (e.g., calibration, CV) to monitor
 carryover from the previous injection. The injection blank consists of the solution in
 Section 7.1.9 fortified with the EIS and NIS for quantitation purposes.

 7.3.7 Stability of solutions – Standard solutions used for quantitative purposes (Sections 7.3.1
 through 7.3.5) should be assayed periodically (e.g., every 6 months) against certified
 standard reference materials (SRMs) from the National Institute of Science and Technology
 (NIST), if available, or certified reference materials from a source that will attest to the
 authenticity and concentration, to assure that the composition and concentrations have not
 changed.

7.4 Sodium iodide/cesium iodide mass calibration solution – 2 mg/mL NaI and 50 µg/mL CsI in (1:1)
 isopropyl alcohol:water (Waters 700000889, or equivalent) or other solution, based on
 manufacturer’s specifications.

7.5 Taurodeoxycholic Acid (TDCA) or Sodium taurodeoxychloate hydrate – (Sigma Aldrich 580221-
 5GM, or equivalent). This compound is used to evaluate the chromatographic program relative to
 the risk of an interference from bile salts in tissue samples. Prepare solution at a concentration of
 100 mg/L in the same solvent as the calibration standards.

Draft Method 1633 - subject to revision 12 August 2021

8.0 Sample collection, preservation, storage, and holding times

8.1 Collect samples in HDPE containers following conventional sampling practices (Reference 5). All
 sample containers must have linerless HDPE or polypropylene caps. Other sample collection
 techniques, or sample volumes may be used, if documented.

8.2 Aqueous samples

 8.2.1 Samples that flow freely are collected as grab samples or in refrigerated bottles using
 automatic sampling equipment. Collect 500 mL of sample (other than leachates) in an
 HDPE bottle. Do not fill the bottle past the shoulder, to allow room for expansion during
 frozen storage.

 Note: Collect at least two aliquots of all aqueous samples to allow sufficient volume for the
 determination of percent solids and for pre-screening analysis. That second aliquot may be
 collected in a smaller sample container (e.g., 250-mL or 125-mL).

 Because the target analytes are known to bind to the interior surface of the sample
 container, the entire aqueous sample that is collected must be prepared and analyzed and
 subsampling avoided whenever possible. Therefore, if a sample volume smaller than 500
 mL is to be used for analysis, collect the sample in an appropriately sized HDPE container.

 8.2.2 Leachate samples from landfills can present significant challenges and therefore only 100
 mL of sample is collected for the analysis. Collect two 100-mL leachate sample aliquots in
 a similar manner as described in Section 8.2.1, using appropriately sized containers.

 8.2.3 Maintain all aqueous samples protected from light at 0 - 6 ºC from the time of collection
 until shipped to the laboratory. Samples must be shipped as soon as practical with
 sufficient ice to maintain the sample temperature below 6 ºC during transport and be
 received by the laboratory within 48 hours of collection. The laboratory must confirm that
 the sample temperature is 0 - 6 ºC upon receipt. Once received by the laboratory, the
 samples must be stored at ≤ -20 ºC until sample preparation.

8.3 Solid (soil, sediment, biosolid), excluding tissue

 8.3.1 Collect samples as grab samples using wide-mouth jars and fill no more than ¾ full (see
 Section 6.1.1.2 for container size and type).

 8.3.2 Maintain solid samples protected from light (in HDPE containers) at 0 - 6 ºC from the time
 of collection until receipt at the laboratory. The laboratory must confirm that the sample
 temperature is 0 - 6 ºC upon receipt. Once received by the laboratory, the samples must be
 stored at ≤ -20 ºC until sample preparation.

8.4 Fish and other tissue samples

 The nature of the tissues of interest may vary by project. Field sampling plans and protocols should
 explicitly state the samples to be collected and if any processing will be conducted in the field (e.g.,
 filleting of whole fish or removal of organs). All field procedures must involve materials and
 equipment that have been shown to be free of PFAS.

 8.4.1 Fish may be cleaned, filleted, or processed in other ways in the field, such that the
 laboratory may expect to receive whole fish, fish fillets, or other tissues for analysis.

Draft Method 1633 - subject to revision 13 August 2021

8.4.2 If whole fish are collected, wrap the fish in aluminum foil or food-grade polyethylene
 tubing, and maintain at 0 - 6 ºC from the time of collection until receipt at the laboratory, to
 a maximum time of 24 hours. If a longer transport time is necessary, freeze the sample
 before shipping. Ideally, fish should be frozen upon collection and shipped to the
 laboratory on dry ice.

 8.4.3 Once received by the laboratory, the samples must be maintained protected from light at
 ≤ -20 ºC until prepared. Store unused samples in HDPE containers or wrapped in
 aluminum foil at ≤ -20 ºC.

8.5 Holding times

 8.5.1 Aqueous samples (including leachates) should be analyzed as soon as possible; however,
 samples may be held in the laboratory for up to 90 days from collection, when stored at
 ≤ -20 ºC and protected from the light. When stored at 0 - 6 ºC and protected from the light,
 aqueous samples may be held for up to 28 days, with the caveat that issues were observed
 with certain perfluorooctane sulfonamide ethanols and perfluorooctane sulfonamidoacetic
 acids after 7 days. These issues are more likely to elevate the observed concentrations of
 other PFAS compounds via the transformation of these precursors if they are present in the
 sample.

 8.5.2 Solid samples (soils and sediments) and tissue samples may be held for up to 90 days, if
 stored by the laboratory in the dark at either 0 - 6 ºC or ≤ -20 ºC, with the caveat that
 samples may need to be extracted as soon as possible if NFDHA is an important analyte.

 8.5.3 Biosolids samples may be held for up to 90 days, if stored by the laboratory in the dark at
 0 - 6 ºC or at -20 ºC. Because microbiological activity in biosolids samples at 0 - 6 ºC may
 lead to production of gases which may cause the sample to be expelled from the container
 when it is opened, as well as producing noxious odors, EPA recommends that samples be
 frozen if they need to be stored for more than a few days before extraction.

 8.5.4 Store sample extracts in the dark at less than 0 - 4 ºC until analyzed. If stored in the dark at
 less than 0 - 4 ºC, sample extracts may be stored for up to 90 days, with the caveat that
 issues were observed for some ether sulfonates after 28 days. These issues may elevate the
 observed concentrations of the ether sulfonates in the extract over time. Samples may need
 to be extracted as soon as possible if NFDHA is an important analyte.

9.0 Quality Control

9.1 Each laboratory that uses this method is required to operate a formal quality assurance program
 (Reference 6). The minimum requirements of this program consist of an initial demonstration of
 laboratory capability, analysis of samples spiked with isotopically labeled compounds to evaluate
 and document data quality, and analysis of standards and blanks as tests of continued performance.
 Laboratory performance is compared to established performance criteria to determine if the results
 of analyses meet the performance characteristics of the method.

 If the method is to be applied to a sample matrix other than water (e.g., soils, biosolids, tissue), the
 appropriate alternative reference matrix (Sections 7.2.2 - 7.2.3) is substituted for the reagent water
 matrix (Section 7.2.1) in all performance tests.

Draft Method 1633 - subject to revision 14 August 2021

9.1.1 The laboratory must make an initial demonstration of the ability to generate acceptable
 precision and recovery with this method. This demonstration is given in Section 9.2.

 9.1.2 In recognition of advances that are occurring in analytical technology, and to overcome
 matrix interferences, the laboratory is permitted certain options to improve separations or
 lower the costs of measurements. These options include alternative extraction,
 concentration, and cleanup procedures, and changes in sample volumes, columns, and
 detectors. Alternative determinative techniques and changes that degrade method
 performance, are not allowed without prior review and approval.

 Note: For additional flexibility to make modifications without prior EPA review, see
 40 CFR Part 136.6.

 9.1.2.1 Each time a modification is made to this method, the laboratory is required to
 repeat the procedure in Section 9.2. If calibration will be affected by the change,
 the instrument must be recalibrated per Section 10. Once the modification is
 demonstrated to produce results equivalent or superior to results produced by this
 method as written, that modification may be used routinely thereafter, so long as
 the other requirements in this method are met (e.g., isotopically labeled
 compound recovery).

 9.1.2.2 The laboratory is required to maintain records of any modifications made to this
 method. These records include the following, at a minimum:

 a) The names, titles, business addresses, and telephone numbers of the analyst(s)
 that performed the analyses and modification, and of the quality control officer
 that witnessed and will verify the analyses and modifications.

 b) A listing of pollutant(s) measured, by name and CAS Registry number.

 c) A narrative stating reason(s) for the modifications (see Section 1.6).

 d) Results from all quality control (QC) tests comparing the modified method to
 this method, including:

 i. Calibration (Section 10)
 ii. Calibration verification (Section 14.3)
 iii. Initial precision and recovery (Section 9.2.1)
 iv. Isotopically labeled compound recovery (Section 9.3)
 v. Analysis of blanks (Section 9.5)
 vi. Accuracy assessment (Section 9.4)

 e) Data that will allow an independent reviewer to validate each determination
 by tracing the instrument output (peak height, area, or other signal) to the final
 result. These data are to include:

 i. Sample numbers and other identifiers
 ii. Extraction dates
 iii. Analysis dates and times
 iv. Analysis sequence/run chronology
 v. Sample weight or volume (Section 11)
 vi. Extract volume prior to each cleanup step (Section 12)

Draft Method 1633 - subject to revision 15 August 2021

vii. Extract volume after each cleanup step (Section 12)
 viii. Final extract volume prior to injection (Section 12)
 ix. Injection volume (Section 13.3)
 x. Dilution data, differentiating between dilution of a sample or extract
 (Section 15.3)
 xi. Instrument
 xii. Column (dimensions, liquid phase, solid support, film thickness, etc.)
 xiii. Operating conditions (temperatures, temperature program, flow rates)
 xiv. Detector (type, operating conditions, etc.)
 xv. Chromatograms, printer tapes, and other recordings of raw data
 xvi. Quantitation reports, data system outputs, and other data to link the raw
 data to the results reported

 9.1.2.3 Alternative columns and column systems – If a column or column system other
 than those specified in this method is used, that column or column system must
 meet all the requirements of this method.

 Note: The use of alternative columns or programs will likely result in a different elution order.

 9.1.3 Analyses of method blanks are required on an on-going basis to demonstrate the extent of
 background contamination in any reagents or equipment used to prepare and analyze field
 samples (Section 4.3). The procedures and criteria for analysis of a method blank are
 described in Section 9.5.

 9.1.4 The laboratory must spike all samples with isotopically labeled compounds to monitor
 method performance. This test is described in Section 9.3. When results of these spikes
 indicate atypical method performance for samples, the samples are diluted to evaluate
 whether the performance issue is caused by the sample matrix. Procedures for dilution are
 given in Section 15.3.

 9.1.5 The laboratory must, on an ongoing basis, demonstrate that the analytical system is in
 control through calibration verification and the analysis of ongoing precision and recovery
 standards (OPR), spiked at low (LLOPR) and mid-level, and blanks. These procedures are
 given in Sections 14.1 through 14.7.

 9.1.6 The laboratory must maintain records to define the quality of data generated. Development
 of accuracy statements is described in Section 9.4.

9.2 Initial Demonstration of Capability

 9.2.1 Initial precision and recovery (IPR) – To establish the ability to generate acceptable
 precision and recovery, the laboratory must perform the following operations for each
 sample matrix type to which the method will be applied by that laboratory.

 9.2.1.1 Extract, concentrate, and analyze four aliquots of the matrix type to be tested
 (Section 7.2.1 through 7.2.3), spiked with 200 µL of the native standard solution
 (Section 7.3.3), 50 µL of the EIS solution (Section 7.3.1), and 50 µL of NIS
 solution (Section 7.3.2). At least one method blank, matching the matrix being
 analyzed, must be prepared with the IPR batch. In the event that more than one
 MB was prepared and analyzed with the IPR batch, all blank results must be
 reported. All sample processing steps that are to be used for processing samples,

Draft Method 1633 - subject to revision 16 August 2021

including preparation and extraction (Sections 11.2 – 11.4), cleanup (Section
 12.0) and concentration (Section 12.0), must be included in this test.

 9.2.1.2 Using results of the set of four analyses, compute the average percent recovery
 (R) of the extracts and the relative standard deviation (RSD) of the concentration
 for each target and EIS compound.

 9.2.1.3 For each native and isotopically labeled compound, compare RSD and %
 recovery with the corresponding limits for initial precision and recovery in Table
 5. If RSD and R for all compounds meet the acceptance criteria, system
 performance is acceptable, and analysis of blanks and samples may begin. If,
 however, any individual RSD exceeds the precision limit or any individual R
 falls outside the range for recovery, system performance is unacceptable for that
 compound. Correct the problem and repeat the test (Section 9.2).

 9.2.2 Method detection limit (MDL) - Each laboratory must also establish MDLs for all the
 analytes using the MDL procedure at 40 CFR Part 136, Appendix B. An MDL
 determination must be performed for all compounds. The minimum level of quantification
 (ML) is then calculated by multiplying the MDL by 3.18 and rounding the result to the
 nearest 1, 2 or 5 x 10n, where n is zero or an integer. Example matrix-specific detection
 limits are listed in Table 6.

9.3 To assess method performance on the sample matrix, the laboratory must spike all samples with the
 isotopically labeled compound standard solution (Section 7.3.1) and all sample extracts with the
 NIS spiking solution (Section 7.3.2).

 9.3.1 Analyze each sample according to the procedures in Sections 11.0 through 16.0.

 9.3.2 Compute the percent recovery of the isotopically labeled compound using the non-extracted
 internal standard method (Section 15.2) and the equation in Section 14.5.2.

 9.3.3 The recovery of each isotopically labeled compound must be within the limits in Tables 9
 and 10 (once the tables are finalized). If the recovery of any compound falls outside of
 these limits, method performance is unacceptable for that compound in that sample.
 Additional cleanup procedures must then be employed to attempt to bring the recovery
 within the normal range. If the recovery cannot be brought within the normal range after
 all cleanup procedures have been employed, water samples are diluted, and smaller
 amounts of soils, biosolids, sediments, and other matrices are prepared and analyzed, per
 Section 15.3.
9.4 Recovery of isotopically labeled compounds from samples must also be assessed and records
 maintained.

 9.4.1 After the analysis of 30 samples of a given matrix type (water, soil, biosolids, tissues, etc.)
 for which the isotopically labeled compounds pass the tests in Section 9.3, compute the R
 and the standard deviation of the percent recovery (S R ) for the isotopically labeled
 compounds only. Express the assessment as a percent recovery interval from R - 2S R to R
 + 2S R for each matrix. For example, if R = 90% and S R = 10% for five analyses of soil, the
 recovery interval is expressed as 70 to 110%.

 9.4.2 Update the accuracy assessment for each isotopically labeled compound in each matrix on a
 regular basis (e.g., after each five to ten new measurements).

Draft Method 1633 - subject to revision 17 August 2021